Septic shock, a complex clinical syndrome that results from a harmful and damaging host response to infection, is the leading cause of mortality in intensive care units. Sepsis develops when the initial appropriate host response to systemic infection becomes dysregulated and over-amplified with an intimate crosstalk between inflammation and coagulation. Among candidates that act as amplifiers of the innate immune response, some belong to the Triggering Receptors Expressed on Myeloid cells (TREM) family [Bouchon, A., et al., 2000; Bleharski, J. R. et al., 2003; Haselmayer, P., et al., 2007; Gibot, S. et al., 2007]. The human TREM gene cluster is located on chromosome 6p21.1 and encodes six different proteins, TREM 1-5 and TLT-1 (TREM-Like Transcript-1). Human TREM-1 (hTREM-1) consists of an extracellular region of 194 amino acid (aa) residues, a membrane spanning region of 29 aa and a short cytoplasmic tail of 5 aa. The extracellular Ig-like domain contains the motif DxGxYxC which corresponds to a V-type Ig-domain. The Ig domain is connected to the transmembrane region by a 60-aa portion containing three N-glycosylation sites. The spanning region contains a Lys residue which forms a salt-bridge with an Asp residue of the transmembrane domain of an ITAM containing protein DAP 12, allowing the association between TREM-1 and its adaptor protein [Bouchon, A., et al., 2000; Kelker, M. S., et Al., 2004; Kelker, M. S. et al., 2004]. Engagement of TREMs triggers a signalling pathway involving ZAP70 and SYK and an ensuing recruitment and tyrosine phosphorylation of adaptor molecules such as GRB2, the activation of PI3K, PLC-γ, ERK-1, -2 and p38 MAPK [Haselmayer, P. et al., 2009; Gibot, S, 2005]. The activation of these pathways that ultimately leads to the activation of transcription factor NF-kB is regulated by CARD9-BCL10-MALT1 [Hara, H. et al., 2007]. Of note, although crystallographic analyses can predict TREM-1 recognition by using antibody-equivalent complementary determining regions (CDR) loops (such as TCRs, CD8 and CTLA-4), its natural ligand has yet to be determined.
Blocking experiments using a TREM-1 fusion protein or using a peptide designed to the CDR3 and the “F” β strand of the extracellular domain of TREM-1 demonstrated a reduced inflammation resulting in improved survival in murine models of endotoxemia and polymicrobial sepsis [Bouchon, A., et al., 2001; Gibot, S. et al., 2006].
The protective effects of modulating TREM-1 signalling are also evident in other models of acute (ischemia-reperfusion, pancreatitis, haemorrhagic shock) or chronic inflammation (inflammatory bowel diseases, inflammatory arthritis). All these studies suggest a role of TREM-1 in amplifying infectious or sterile inflammation.
In addition to TREM-1, the TREM gene cluster includes TREM-like Transcript 1 (TLT-1). TLT-1 is abundant, exclusively expressed on platelets and megakaryocytes, and is sequestered in the platelet α granules. Upon platelet activation, TLT-1 is translocated to the platelet surface [Washington, A. V. et al., 2004]. TLT-1 contains a v-set Ig type-extracellular domain, a transmembrane region and a cytoplasmic tail that comprises an immunoreceptor tyrosine-based inhibitory motif (ITIM) and a polyproline-rich domain. Unlike other TREM family members, TLT-1 does not couple to the DAP 12 activating chain whereas it has been shown to enhance Ca++ signalling in rat basophilic leukemia (RBL) cells, suggesting TLT-1 is a co-activating receptor [Barrow, A. D. et al., 2004].
The specificity of TLT-1 expression on platelets suggested that it should play a unique role in haemostasis and/or thrombosis. Indeed, it has been suggested that the modulation of TLT-1 may have several potential in modulating platelet function, thus preventing inflammatory-associated hypercoagulation (but not directly inflammation) or platelet-associated disorder (e.g. bleeding or clotting disorder) [Washington A V. et al., 2009]. This modulation could be mediated in a direct (by interfering with a TLT-1-ligand) or indirect way (by modulating the TLT-1 intracellular pathway).
The inventors report for the first time herein that TLT-1 and TLT-1 derived peptides exhibit anti-inflammatory properties by specifically inhibiting TREM-1 activity. Such peptides are able to dampen TREM-1 signalling and thus behave as naturally occurring TREM-1 inhibitors. They further demonstrate that the same peptides, as a consequence of the inhibition of TREM-1 and its intracellular pathway, also modulate in vivo the proinflammatory cascade triggered by infection, thus inhibiting TREM-1-associated hyper-responsiveness and ensuing organ damages and death during sepsis.